Myosin is a major contractile protein present in all eukaryotic cells which plays a role in cell proliferation and specialized cellular function. We have been cloning cDNAs encoding nonmuscle myosin heavy chains (MHCs) in human T-cells to study the role of MHCs in cell function. We have already obtained a number of cDNA clones encoding two different human nonmuscle MHCs (NMMECs) designated NMMHC-A and NMMHC-B. To fill in a gap in the sequence of NMMHC-A and to complete the nucleotide sequence of the remainder of NMMHCB, we screened a Stratagene lambda ZAP II T-cell library and Clontech lambda gtl0 T-cell library with cDNA probes. Two cDNA clones (Z801, Z802) that completed the gap sequence in NMMHC-A were isolated (Simons et al., Circ. Res. 69: 530-539, 1991). We also isolated, 5 cDNA clones (HB5.12, HB3.18, HB3.2, HB3.21 and HB4.5) which almost completed the sequence of NMMHC-B. The amino acid sequences of human NMMHC-B was more similar to that of the chicken NMMHC-B (95% identity) as compared to human NMMHC-A (77% identity). The amino acid sequence of chicken smooth muscle myosin was homologous to human NMMHCs, but there was no significant difference among human.NMMHC-A, B and chicken smooth muscle myosin (% identity in amino acids: 74%,.75%, respectively). We constructed two peptides from the C-terminal sequence of the two isoforms to generate specific antibodies to distinguish NMMHC-A protein from B. ELISA and immunoblot tests showed that our antibodies were able to distinguish two isoforms. We also obtained an antibody that recognizes both NMMHC-A and B by the immunization of a peptide in the head region of NMMHC-B. To examine the function of NMMHCs in T-cells, we are planning to use antisense oligonucleotides from the 5' untranslated region which include the first ATG.